Figure 5.

Inhibition of PKA substrates and tyrosine phosphorylations by TSP but not by MJ33 was prevented by D-penicillamine (PEN). Percoll-selected human spermatozoa were capacitated with 10% FCSu in BWW medium with or without 10 μM TSP or MJ33 in the presence or absence of 2 mM D-PEN at 37°C for 4 h. Sperm suspensions were supplemented with SDS-PAGE sample buffer and solubilized proteins from 0.5 × 10⁶ spermatozoa were loaded in each well, electrophoresed, electrotransferred and immunoblotted using the (A) anti-P-PKA substrates (P-PKA-S; 1:1000 dilution) or (B) the anti-P-Tyrosine antibody (1:10 000). Equal loading control was determined by using an anti-α-tubulin antibody (1:10 000). The immunoblots are representative of four experiments (n = 4) different donors. Relative intensity of P-Tyr or P-PKA-S was normalized to the relative intensity of tubulin of each sample and expressed as mean ± SEM. *P < 0.05 versus non-capacitated controls or sperm samples capacitated in the presence of MJ33 (n = 4).